We propose to develop biomimetic nanospheres and the necessary instrumentation for their analysis, to create an integrated platform capable of optimal protease assays. This platform will have several significant advantages, which include the use of full-length substrates, the ability to multiplex assays, and, most importantly, the abilty to provide protein substrates as saturating concentrations for protease reactions, which enables the determination of key kinetic parameters. These properties represent a near ideal combination for a protease assay platform and are not matched by any other assay approach available today. The development of an ideal protease assay platform has great medical significance, as there are hundreds of proteases directly involved in many important human diseases. For example, proteases cleave protein targets (substrates) important in the progression of disease, like viral processing by the flaviviridae, toxin cleavage of proteins, and digestion of the extracellular matrix during cancer metastasis. The need for ideal protease assays is further driven by the need for protease inhibitors (5-10% of drug discovery targets proteases). The combination of the number of proteases of scientific interest and their great pharmaceutical value makes the protease assay among the most important assays in medical science. We will create a near ideal protease assay by implementing on the nanoscale our surface based assays that have been successful on multiplex microsphere arrays. Nanoparticle protease assays will use cleavable fluorescent fusion protein substrates to detect protease activity by monitoring the loss of fluorescence from the microsphere surface via flow cytometry. In our proposed platform, particle presence, particle type, and fluorescence changes will be detected using a multiparameter, high-sensitivity flow cytometer optimized for multiplex fluorescent nanosphere array analysis. Successful development of our platform will be demonstrated via in vitro assays against toxin and viral proteases. These protease assays will be used to derive kinetic parameters, multiplex assays, and measure inhibitor activity. These goals will require the development of multiplexed nanosphere arrays bearing biomimetic surfaces, the creation of a high sensitivity flow cytometer with attoliter analytical volumes, and the demonstration of protease assays using these integrated technologies. If successful, our protease assay format will be simple to perform, multiplexable, provide key kinetic parameters (kcat, Km, and Ki), and display large protein substrates in a biologically relevant fashion to enable contributions from large protease to protein interactions (e.g. exosites) to be evaluated. Our platform would have clear value by providing an optimal protease assay for use in biomedical research. Beyond this, the novel nanoparticles and flow cytometry technology will be useful for a variety of analytical studies in addition to those pursued here. Thus, beyond an improved protease assay, the technology developed will have widespread general benefit to the scientific and biomedical communities.